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The development of the venous pole of the heart

Mommersteeg, M.T.M.

Link to publication

Citation for published version (APA):Mommersteeg, M. T. M. (2009). The development of the venous pole of the heart.

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5Molecular pathway for the localized

formation of the sinoatrial node

Mathilda T.M. Mommersteeg, Willem M.H. Hoogaars, Owen W.J. Prall,

Corrie de Gier-de Vries, Cornelia Wiese, Danielle E.W. Clout,

Virginia E. Papaioannou, Nigel A. Brown, Richard P. Harvey,

Antoon F.M. Moorman, Vincent M. Christoffels

Circulation Research 2007;100;354-362

Chapter

thesis_arial_73:Opmaak 1 26-7-2009 20:49 Pagina 81


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Abstract

The sinoatrial node, which resides at the junction of the right atrium and the superior

caval vein, contains specialized myocardial cells that initiate the heart beat. Despite

this fundamental role in heart function, the embryonic origin and mechanisms of lo-

calized formation of the sinoatrial node have not been defined. Here we show that

subsequent to the formation of the Nkx2-5-positive heart tube, cells bordering the in-

flow tract of the heart tube give rise to the Nkx2-5-negative myocardial cells of the

sinoatrial node and the sinus horns. Using genetic models, we show that as the my-

ocardium of the heart tube matures, Nkx2-5 suppresses the pacemaker channel gene

Hcn4 and T-box transcription factor gene Tbx3, thereby enforcing a progressive con-

finement of their expression to the forming Nkx2-5-negative sinoatrial node and sinus

horns. Thus, Nkx2-5 is essential for establishing a gene expression border between

the atrium and sinoatrial node. Tbx3 was found to suppress chamber differentiation,

providing an additional mechanism by which the Tbx3-positive sinoatrial node is

shielded from differentiating into atrial myocardium. Pitx2c-deficient fetuses form

sinoatrial nodes with indistinguishable molecular signatures at both the right and left

sinuatrial junction, indicating that Pitx2c functions within the left/right pathway to sup-

press a default program for sinoatrial node formation on the left. Our molecular path-

way provides a mechanism for how pacemaker activity becomes progressively

relegated to the most recently added components of the venous pole of the heart,

and, ultimately, to the junction of the right atrium and superior caval vein.

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5

Chapter 5 - Nkx2-5, Pitx2c and Tbx3 in SAN formation


Introduction

The sinoatrial node (SAN) is the pacemaker of the heart that initiates the heartbeat

and controls its rate of contraction. It is a specialized myocardial structure positioned

at the junction of the right atrium and the superior caval vein. Disease, ageing, or

gene defects may cause SAN dysfunction (sick sinus syndrome), a common cardiac

disorder that currently requires the implantation of a pacemaker. SAN function de-

pends on a complex tissue architecture and the expression and function of a set of

ion-channel, sarcomeric and gap junction proteins expressed at levels distinct from

the atrial working myocardium.1,2 Hyperpolarization-activated pacemaker channel

Hcn4 is expressed in the SAN and plays an important role in pacemaker activity in

vivo.2,3 Furthermore, the SAN expresses very low levels of Connexin 40 (Cx40) and

Cx43 gap-junctional subunits that are essential for the fast conduction of the electrical

impulse in the atrial and ventricular working myocardium and ventricular conduction

system.4,5 The T-box transcription factor, Tbx3, which, when mutated, causes ulnar-

mammary syndrome of congenital malformation in human,6 is expressed in the de-

veloping nodal tissues of the heart of mammals and chicken and is able to suppress

promoter activity of atrial chamber myocardial genes including natriuretic peptide pre-

cursor type A (Nppa, encoding atrial natriuretic factor) and Cx40 in cell culture.7

Despite its fundamental role in heart function, the embryonic origin of the

SAN and the mechanisms of its patterning and formation are still enigmatic. In the

present study we investigated the spatio-temporal patterns of genes relevant to the

developing inflow tract, SAN and atrium and provide evidence for roles for Nkx2-5

(NK2 transcription factor related, locus 5 [Drosophila]), Pitx2c (paired-like home-

odomain transcription factor 2), and Tbx3 in the patterning and formation of the SAN.

Materials and Methods

Mice

The R26R,8 Nkx2-5-,9 Nkx2-5ires-Cre,10 Pitx2c-,11 and Tbx3- 12 transgenic mouse lines

have been described previously. A cDNA fragment encoding the full-length human

TBX3 protein13 was cloned in a construct containing a 5-kb regulatory fragment of

the murine -Mhc gene and the human growth hormone pA signal,14 kindly provided

by Dr. J. Robbins (Children’s Hospital Research Foundation, Cincinnati, Ohio). As a

control, a cDNA fragment encoding TBX3 with a point mutation in the T-box DNA

binding region (L143P)13 was cloned in the same construct. Vector sequences of

these constructs were removed and the DNA was microinjected into nuclei of FVB


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zygotes, which were subsequently implanted into fosters to generate transgenic em-

bryos. At embryonic day 9.5 (E9.5), embryos were isolated for analysis. Embryos and

fetuses were dissected in PBS and fixed in 4% paraformaldehyde overnight. Amnion

or tail biopsy genomic DNA was used for PCR assays to detect the Cre, lacZ, GFP

or TBX3 transgenes.

β-galactosidase activity detection and immunohistochemical analyses

Detection of β-galactosidase activity on 20 μm cryostat sections was performed as

described.15 For immunohistochemistry on 10 μm embryo sections, the following pri-

mary antibodies were used: polyclonal antibody against cardiac troponin I (cTnI)

(1:1000; Hytest Ltd); polyclonal antibody against Nkx2-5 (1:250; Santa Cruz Biotech-

nology); polyclonal antibody against Hcn4 (1:200; Chemicon); polyclonal antibody

against Cx40 (1:200; Chemicon); monoclonal antibody against desmin (1:50; Mono-

san).

Non-radioactive in situ hybridization

Non-radioactive in situ hybridization of 12 μm embryo sections was performed as de-

scribed.16 The probes for Isl117 and Hcn418 were kindly provided by S. Evans (Skaggs

School of Pharmacy, University of California, San Diego) and B. Santoro (Center for

Neurobiology and Behavior, Columbia University, New York). Other probes have been

described previously.16 All patterns in wild-type embryos were observed in at least 3

independent embryos.

Three-dimensional reconstructions

Three-dimensional visualization and geometry reconstruction of patterns of gene ex-

pression determined by in situ hybridization was performed as described previously.19

Files with reconstructions are available upon request.

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Figure 1. Spatial and temporal patterns of SAN and atrial genes indicate progressive patterning and for-

mation of the primordial SAN. A, Embryos stained whole mount show expression of Nppa in the atria and ven-

tricles at E9.5 and E10.5. The black line indicates the level of sectioning in panel B. B, Three sister sections of an

E9.5 heart show coexpression of cTnI, Nkx2-5 and Hcn4 in the inflow tract. Three sister sections of an E10.5 heart

show expression of Hcn4 selectively in the sinus horns, and of Nkx2-5 excluded from the sinus horns (black arrow-

heads indicate the sinus horn myocardium). Three sister sagittal sections of an E10.5 heart show some overlap in

expression of Hcn4 and Nkx2-5, also in the Nkx2-5-positive cells of the second heart field in the dorsal mesocardium

(red arrowheads). Black arrowheads indicate the sinus horn myocardium. C, Whole mount Nppa stained E9.5

embryo indicating plane of sectioning (black line) and Cx40 expression in the inflow tract fated to become atrium.

Two pairs of sister sections of an E10.5 embryo showing the expression of Nppa and Cx40 in the atria but not in

the sinus horns, and Tbx3 expression in a right-sided subdomain of the Cx40-negative sinus horns (*). a indicates

atrium; Cra, cranial; Cau, caudal; d, dorsal; dm, dorsal mesocardium; ift, inflow tract; l/rsh, left/right sinus horn; l/ra,

left/right atrium; l/rv, left/right ventricle; v, ventral . Bars represent 100 μm.


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5Figure 2. Expression and lineage analysis of the venous pole. A, Three-Dimensional reconstructions of E14.5

wild-type embryos showing the lumen (orange) of the heart from dorsal (top panels) and from the right side (bottom

panels). In red, Tbx3-positive myocardium; in gray, Cx40-negative myocardium. The Tbx3 expression pattern is

confined to the Cx40-negative myocardium. B, sister sections of a wild-type E11.5 embryo double-stained with an-

tibodies against desmin (blue) and Hcn4 or Nkx2-5 (pink). Nuclei are stained with SYTOX Green (green). C and D,

Sister sections of E14.5 wild-type embryos showing the mutually exclusive expression patterns in the SAN and

right atrium. E, Schematic representation of the patterns of expression until E9.5 (left) and between E9.5 and E14.5

(middle). Dotted lines represent borders of the expression domains of Nkx2-5 and Pitx2c. Except for the yellow-co-

lored mesenchyme, only myocardium is depicted (see legend in right panel). san indicates sinoatrial node; l/rsh,

left/right sinus horn; l/ra, left/right atrium; avc, atrioventricular canal; l/rv, left/right ventricle; vv, venous valves; ift,

inflow tract; l/raa, left/right atrial appendage; as, atrial septum; pv, pulmonary vein. Bars represent 100 μm.


Results

Expression and genetic lineage analysis reveal a mechanism of SAN development

To investigate formation of the SAN in development, we assessed the expression

patterns of Hcn4, a SAN marker1; Nppa and Cx40, atrial chamber myocardial mark-

ers4; Tbx3, a developing conduction system marker and putative repressor of Nppa

and Cx407; Nkx2-5, a key cardiac homeobox gene expressed in the first and second

heart fields20 and essential for the activation of Cx40 and Nppa,21,22 and cTnI, desmin

and myosin light chain 2a (Mlc2a), which mark all differentiated myocardium. “Inflow

tract” refers to the myocardial intake at the caudal end of the heart tube until E9.5.

“Venous pole” refers to the myocardial components of the systemic venous return

that are added to the heart from E9.5 onward, including the SAN (this study), venous

layer of the bilayered venous valves, and left and right sinus horns.16

The E9.5 heart tube coexpressed myocardial marker genes and Nkx2-5. The

inflow tract in addition expressed Hcn4 in a pattern that initially overlaps that of

Nkx2-5 (Figure 1A and 1B). Expression of Cx40 had been initiated at this stage in

the Nkx2-5-positive inflow tract (Figure 1B and 1C). After E9.5, Nkx2-5-negative

myocardial sinus horns are formed from cardiac progenitor cells adjacent to the inflow

tract.16 At E10.5, Cx40 and Nppa were expressed in the Nkx2-5-positive myocytes,

which at this stage have contributed to the atrial chambers and atrial layer of the ve-

nous valves. Hcn4 expression was down-regulated in this myocardium to become

confined to the newly formed Nkx2-5-negative venous pole (Figure 1B and 1C). Thus,

an early pattern in which Nkx2-5 and Cx40 expression overlaps that of Hcn4 resolves

into a mutually exclusive pattern by E10.5.

From E10.5 on, a thickening of myocardial cells was formed in the right sinus

horn bordering but exclusive of the Nkx2-5/Cx40/Nppa-positive atrial cells, corre-

sponding to the primordial SAN23 (Figure 1C). This structure also expressed Tbx3.

Three-dimensional reconstructions show that the expression domains of Tbx3 and

of Cx40 were mutually exclusive (Figure 2A). At the atrial side, the Tbx3 expression

domain bordered the Cx40-positive domain, but neither Tbx3 nor Cx40 were ex-

pressed in the myocardium of the sinus horns (Figure 1C and 2A). Together, these

data indicate the existence of 3 bordering domains at the sinuatrial junction, an atrial

domain, a SAN domain and a sinus horn domain (Figure 2B through 2E).

To address whether the Nkx2-5-negative SAN primordium is derived from

Nkx2-5-positive inflow tract myocardium, which subsequently loses Nkx2-5 expres-

sion, or, like the sinus horns, from adjacent Nkx2-5-negative mesenchyme,16 we

crossed Nkx2-5ires-Cre mice10 with R26R mice8 to reveal all cells derived from Nkx2-5-

positive cells. These data showed that the Tbx3-positive, Cx40-negative SAN pri-


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mordium was devoid of Nkx2-5-Cre-positive cells, and therefore never expressed

Nkx2-5 (Figure 3A and 3C). We conclude that the primordial SAN myocardium is

formed de novo from Nkx2-5-negative cells.

LIM homeodomain transcription factor islet-1 (Isl1) is reported to be a marker

of the second heart field precursors that is switched off when these mesenchymal

precursors differentiate to myocardium.17 However, probably because of the half-life

of its transcripts, Isl1 expression remains detectable shortly after differentiation of the

precursors into myocardium.16 Isl1 expression was still observed in the Hcn4-positive

primordial SAN (Figure 3B), providing support for de novo differentiation of the SAN

from second heart field progenitors.

Nkx2-5 suppresses Hcn4 and Tbx3 and establishes the sinoatrial boundary

The boundary between the SAN and atrial myocardium could be established at least

in part if Nkx2-5 acted as a repressor of Hcn4 and Tbx3. We therefore tested the ex-

pression of Hcn4 and Tbx3 in Nkx2-5-deficient mice. In these embryos, heart devel-

opment is arrested during looping, chamber formation does not occur, and Cx40 and

Nppa are not activated.21,22 We found a marked ectopic expression of both Hcn4 and

Tbx3 throughout the heart tubes of Nkx2-5-deficient embryos (Figure 4A), showing

that Nkx2-5 is required to repress these genes in the heart tube. These observations

indicate a critical role of Nkx2-5 in the confinement of Hcn4 and Tbx3 to the Nkx2-5-

negative venous pole domain (Figure 4C).

Because Nkx2-5 mutant mice die at E10, this model does not allow the analy-

sis of the SAN region in a more advanced state of development. We investigated

fetal mice compound heterozygous for the Nkx2-5ires-Cre allele and for a null allele.

These mice express ~25% of the normal level of Nkx2-5 expression in wild-type, be-

cause the ires-Cre insert in the 3' untranslated region of the Nkx2-5 gene creates a

hypomorphic allele (O.P, R.P.H., submitted manuscript, 2006). Nkx2-5ires-Cre / - mice

die shortly after birth showing a range of cardiac malformations, including atrial and

ventricular septal defects, much more severe than haploinsufficient mice. As controls,

Nkx2-5ires-Cre / + mice were used which appeared completely normal. In Nkx2-5ires-Cre / -

E14.5 fetuses, the Hcn4 and Tbx3 expression domains of the SAN were much

broader and extended into the atrium (Figure 4B). Thus, the expression border be-

tween the SAN and atrium was blurred. Cx40 expression was reduced in these

hearts, consistent with the notion that Nkx2-5 is required for Cx40 expression,22 but

was particularly weak in the enlarged Hcn4 and Tbx3 domain around the SAN (Figure

4B). These observations indicate a critical role of Nkx2-5 in the regulation and main-

tenance of the atrial chamber gene program at the border with the SAN domain (Fig-

ure 4C).

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Figure 3. SAN forms from Nkx2-5-negative precursore. A, E12.5 Nkx2-5ires-Cre/R26R embryo stained with5-

bromo-4-chloro-3-indolyl β-D-galactoside (Xgal) (n=3) and the corresponding area in an E12.5 wild-type embryo

stained for Cx40 and Tbx3. The black box in the left panel depicts the area enlarged in the right panels. Arrows

depict the Xgal and Cx40-negative, Tbx3-positive SAN area. B, Sister sections of the SAN area of an E10.5 wild-

type embryo. Black arrows, overlap of cTnI, Hcn4 and Isl1 expression in the Nkx2-5-negative primordial SAN. C,

Simplified scheme of the development of the venous pole and the SAN. At E9.5 the inflow tract myocardium ex-

presses both Hcn4 and Nkx2-5. After E9.5, the Hcn4-positive area becomes gradually confined to the Nkx2-5-

negative myocardial venous pole that is recruited from adjacent non-cardiac progenitor cells. Black arrows indicate

recruitment of mesenchyme into the Nkx2-5-negative myocardial venous pole lineage. Part of the Nkx2-5-negative

myocardium initiates Tbx3 expression and thickens to form the SAN primordium. After E14.5 Hcn4 expression be-

comes confined to the Tbx3-positive SAN, whereas in the sinus horn myocardium Cx40 is up-regulated. ra indicates

right atrium; rsh, right sinus horn; rvv, right venous valve. Bars represent 100 μm.


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5Figure 4. Nkx2-5 represses Hcn4 and Tbx3 and is required to establish the atrial-SAN boundary. A, E9.0

Nkx2-5+/- and Nkx2-5-/- embryos stained whole mount for Tbx3 (n=3) and on sister sections for myocardial marker

Mlc2a and for Hcn4 (n=3). Arrow depicts the upregulation of Tbx3 and Hcn4 throughout the entire heart in absence

of Nkx2-5. B, Sister sections of Nkx2-5ires-Cre/+ (control; n=7) and Nkx2-5ires-Cre/- (hypomorphic; n=9) embryos of E14.5.

Arrows depict the expression border of SAN and atrial genes. C, Scheme depicting roles and genetic interactions

found in this study. Factors present in a given compartment are depicted in black, factors absent from a compartment

in gray. ev indicates embryonic ventricle; ift, inflow tract; lsh, left sinus horn; ra, right atrium; san, sinoatrial node;

vv, venous valves. Bars represent 100 μm.


Tbx3 suppresses chamber myocardial differentiation and gene expression

Our analysis revealed that Tbx3 could be involved in the suppression of atrial cham-

ber differentiation and repression of chamber gene expression, thus contributing to

the chamber-negative phenotype of the SAN. We analysed Tbx3 mutant embryos

which die from between E11-E13.12 Embryos that clearly survived until E11.5 had

formed a primordial SAN-like structure (Figure 5A). Moreover, Nkx2-5, Cx40 and

Nppa were not ectopically activated in this primordium, and Hcn4 expression was

maintained (Figure 5A). Notably, because Nkx2-5 is strictly required for Nppa and

Cx40 gene activity,21,22 maintenance of the Nkx2-5 boundary at the junction between

the atrium and SAN/venous pole at this developmental stage provides a likely expla-

nation for the maintenance of the SAN gene expression signature in the Tbx3 mu-

tants.

Nonetheless, Tbx3 may provide a tuning mechanism to ensure stabilization

of SAN gene expression, in particular, to ensure the absence of the atrial differentia-

tion program. To investigate whether Tbx3 is able to suppress chamber myocardial

differentiation and Nppa and Cx40 in vivo, transgenic embryos were generated that

express the human Tbx3 cDNA under the control of the -Mhc promoter, which drives

expression in the tubular heart prior to the differentiation of chamber myocardium.14,24

To evaluate possible nonspecific effects of Tbx3 overexpression, eg, by competing

for limiting cofactors, we also generated embryos that express Tbx3-LP, which con-

tains an L to P substitution at position 143 in the T-box domain that has been found

in ulnar-mammary syndrome patients.6 Tbx3-LP and Tbx3 protein showed compara-

ble stability in transfected cells,7,13 but Tbx3-LP is not able to bind to its DNA binding

sites.7 Transgenic embryos were analyzed at E9.5, after the initiation of chamber dif-

ferentiation and Nppa and Cx40 expression. These embryos were smaller but not

obviously abnormal with respect to general body patterning (Figure 5B). Hearts of

these embryos were linear, or looped to some extent, and partially or completely

failed to form chambers (Figure 5B and 5C). Section in situ hybridization showed that

Nppa and Cx40 were not expressed in these hearts, indicating that their expression

was suppressed by Tbx3 (Figure 4C and 5C). However, Hcn4 was not precociously

activated in the Tbx3-overexpressing hearts (data not shown), supporting the data

obtained in the Tbx3 mutant suggesting that Tbx3 does not directly regulate Hcn4

(Figure 4C). Tbx3-LP embryos were normal, and even though Tbx3-LP was robustly

expressed in the hearts, the morphology and the expression of Cx40 and Nppa ex-

pression were not affected (Figure 5C). We conclude that Tbx3 is able to specifically

block chamber myocardial differentiation and the expression of Nppa and Cx40 in

vivo in a DNA-binding dependent manner.


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Pitx2c suppresses SAN formation in the left side of the venous pole

The Pitx2 homeobox factor is essential for late aspects of left-right asymmetric mor-

phogenesis.25 Pitx2c-deficient mutant mice have right isomerism of the atria and ve-

nous pole.11 In situ hybridization showed that the primordial SAN does not express

Pitx2c (Figure 6A). We investigated whether Pitx2c is also involved in the placement

of the primordial SAN. Pitx2c mutant embryos of E9.5, shortly before the primordial

SAN is formed and the asymmetry of the sinus horns becomes apparent,26 have ap-

parently normal inflow tracts (not shown). However, after E10.5, shortly after initiation

of the formation of the primordial SAN, we noted that the atria had 2 sets of venous

valves, and 2 short sinus horns, indicating right isomerism of the left venous pole

(Figure 6B). Moreover, a second SAN primordium was invariably observed at the left

side. This left-sided SAN primordium showed an appropriate Hcn4-positive, Cx40

negative SAN gene program. At E15.5, the left-sided SAN contained the typical nodal

artery, and the gene expression profile of this SAN was indistinguishable from that of

the right sided SAN in the mutant, and of the SANs of wild-type littermates. We con-

clude that the default morphogenetic pathway of the venous pole leads to the forma-

tion of a right-sided sinus horn, SAN and venous valves, and that the Pitx2c pathway

suppresses SAN formation.

Atrialization of the venous pole

From the above we, conclude that after establishment of an Nkx2-5-expressing heart

tube, the sinus horn myocardium and SAN are formed from cardiac field cells via a

novel, Nkx2-5-independent pathway and run a nodal gene program. However, from

E14.5 onward, expression of Cx40 was up-regulated in the sinus horn myocardium,

whereas that of Hcn4 was gradually lost from the sinus horn myocardium (Figure 7A

through 7D). Furthermore, the sinus horn myocardium gradually became positive for

Nkx2-5 expression (Figure 7C and 7D). Moreover, regions within the SAN initiated

Nkx2-5 expression, which now for the first time overlapped with Tbx3 expression (Fig-

ure 7E through 7G). However, Tbx3 and Hcn4 were not downregulated and the SAN

remained free of Cx40 expression (Figure 7F and 7G). Taken together, the data sug-

gest that in the fetal period, the venous pole myocardium obtains an atrial gene pro-

gram, except for the SAN, which, despite the up-regulation of Nkx2-5, still runs the

Tbx3/Hcn4-positive, Cx40-negative nodal gene program (Figure 3C).

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Figure 5. Tbx3 suppresses chamber differentiation and Nppa and Cx40 gene expression in vivo. A, Sister

sections of E11.5 Tbx3+/+ and Tbx3-/- littermates. Arrow depicts the absence of Cx40 and presence of Hcn4 in the

SAN primordium of both the wild-type and Tbx3-deficient embryo (n=2). A sinoatrial node primordium was observed

in 4 of 4 mutant embryos. B, E9.5 βMhc-hTBX3 and wild-type littermate. C, Serial sections of E9.5 wild-type, βMhc-

hTbx3 and βMhc-hTBX3LP embryos and a whole-mount picture of the heart region of the embryo showing the ab-

sence of Nppa and Cx40 in the Tbx3-overexpressing βMhc-hTBX3 embryo. Of βMhc-hTBX3 embryos (n=6), 3 had

linear heart tubes, 1 had a looped heart tube but no chambers, and 2 showed looping and thin-walled chambers.

Hearts of all βMhc-hTBX3LP embryos (n=5) were normal. Hearts of all wild-type littermates examined (n=30)

showed normal looping and chamber formation. avc indicates atrioventricular canal; ht, heart; lsh/rsh, left/right

sinus horn; lv, left ventricle; san, sinoatrial node. Bars represent 100 μm.


Discussion

In this study, we demonstrate that the SAN initially forms from Nkx2-5-negative pre-

cursors, and remains largely free of Nkx2-5 expression until the onset of a second

phase of atrial myogenesis in the venous pole beginning at E14.5. Nkx2-5 is a cardiac

homeobox gene critical for heart development in all species examined thus far and

is at the core of transcriptional regulatory mechanisms that control gene expression

and differentiation in the heart.21 Heterozygous mutations in Nkx2-5 in humans cause

congenital atrioventricular conduction system defects.27 Moreover, the atrioventricular

node, which expresses relatively high levels of Nkx2-5, is hypoplastic in heterozygous

mutant mice.28 Therefore, although the SAN and the atrioventricular node share many

phenotypic features, the differential expression of Nkx2-5 is a fundamental difference

between the 2 nodes, implying that distinct regulatory mechanisms control their for-

mation and function.

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Figure 6. Pitx2c represses SAN gene program and morphogenesis at the left side. A, Sister sections of an

E13.5 wild-type embryo stained for Nkx2-5 and Pitx2c expression. Arrows indicate lack of expression of Pitx2c in

Nkx2-5-negative SAN. B, Sister sections of an E11.5 (n=5) and an E14.5 (n=11) Pitx2c-/- embryo. Arrows indicate

an Hcn4/Tbx3-positive, Nkx2-5/Cx40-negative SAN at both right and left side. avc indicates atrioventricular canal;

la/ra, left/right atrium; lsh/rsh, left/right sinus horn; lv, left ventricle; san, sinoatrial node. Bars represent 100 μm.


5


Early localization of pacemaker activity and patterning of the venous pole

All embryonic cardiomyocytes of the early developing heart possess intrinsic molec-

ular pacemaker activity, which is followed by contractility slightly later. However, dom-

inant pacemaker activity is always found at the intake (inflow tract, venous pole) of

the developing heart tube.29,30 Hcn4 is required for pacemaker activity in mouse

embryos, whereas humans with a mutation in HCN4 have bradycardia.2,3 The steep

caudo-cranial expression gradient of Hcn4 observed from E7.5 in the cardiac cres-

cent31 and subsequent expression in the entire venous pole (SAN and sinus horns;

Figures 1B, 2E, and 3C) may well explain the observation that dominant pacemaker

activity is always localized at the intake.

The embryonic heart tube elongates by recruitment of cardiac field cells to

the heart tube and inflow complex.20 In the inflow tract region, the atria are formed

first, followed after E9.5 by the sinus horns16 and the primordial SAN (this study). Ex-

pression of Hcn4 initially overlaps with that of Nkx2-5 but subsequently becomes re-

stricted to the newly recruited Nkx2-5-negative venous pole components, indicating

that Hcn4 expression is extinguished in the Nkx2-5-positive myocardium (Figures 2E

and 3C), which is fated to form the atria and atrial layer of the venous valves.16 In

line with these patterns of expression, Nkx2-5-deficient embryos showed a dramatic

ectopic expression of Hcn4 in the heart tube, explaining previous observations that

a large fraction of Nkx2-5-deficient embryos initiate beating from the embryonic ven-

tricular region rather than from the inflow tract.22 Together, these data indicate that

Hcn4 expression is initiated by unknown factors in newly formed venous pole my-

ocardium and that as the heart matures Nkx2-5 represses Hcn4, thereby confining

its expression to the Nkx2-5-negative venous pole (Figures 3C and 4C). This repres-

sive activity for Nkx2-5 on Hcn4 provides a mechanism for the progressively shift of

Hcn4 expression as well as pacemaker activity to the newly formed venous pole com-

ponents in normal embryos (Figures 3C and 4C). This mechanism provides an ex-

planation for the longstanding observation that during heart development dominant

pacemaker activity is always localized at the caudal end (intake) of the heart.29,30

Patterning and formation of the SAN

Nkx2-5-deficient embryos showed ectopic expression of Hcn4 and Tbx3 in the whole

heart tube. Furthermore, in Nkx2-5 hypomorphic fetuses the otherwise sharp expres-

sion boundary between the atria and SAN was blurred. The expression of Tbx3 and

Hcn4 extended into the right atrium, whereas Cx40 expression was specifically down-

regulated in the extended Tbx3-positive area. These observations indicate that

Nkx2-5 is required to establish the boundary between the atria and SAN and, in a

dose-dependent manner, to prevent the SAN phenotype from invading the atria, or

the atrial phenotype invading the SAN (Figure 3C and 4C).


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5Figure 7. Atrialization of the venous pole. A, Sister sections of an E14.5 heart. The Tbx3-negative left sinus horn

coexpresses Cx40 and Hcn4 (red arrows). B, Section of an E17.5 wild type heart showing the downregulation of

Hcn4 in the sinus horns, whereby its expression is confined to the SAN. C and D, Sister sections of an E17.5 wild-

type heart. Black arrows show the up-regulation of Nkx2-5 and Cx40 in the sinus horns. The red arrows indicate

also some up-regulation of Nkx2-5 in the Cx40-negative SAN region. E, E15.5 (n=2) and E17.5 (n=2) Nkx2-5ires-

Cre/R26R embryos double-stained for Xgal and cTnI. Note the increase in recombination in the SAN between these

stages. F, Sister sections of an E17.5 heart show upregulation of Nkx2-5 mRNA in the Tbx3-positive SAN (red ar-

rows). G, Sister sections of an E17.5 heart show the presence of Nkx2-5 in the Cx40-negative SAN (white arrows).

cg indicates cardiac ganglion; lsh/rsh, left/right sinus horn; na, nodal artery; ra, right atrium; san, sinoatrial node.

Bars represent 100 μm.


A next step involves asymmetrical gene expression and morphogenesis after

E9.5, which leads to the formation of a Tbx3-expressing SAN primordium at the right

sinoatrial junction. Human patients and iv/iv mice with right isomerism have 2

sinoatrial nodes, whereas in patients and mice with left isomerism the sinoatrial node

is hypoplastic or absent.32,33 Therefore, the SAN is an integral component of the right

side-specific morphogenesis program that is repressed by Pitx2c. We found that

Pitx2c-deficient embryos form 2 SANs running the SAN signature gene program.

Therefore, the complete right-sided morphogenesis program, including Tbx3 expres-

sion, is the default program, which in the left sinoatrial region is masked and con-

verted to a left-sided program without a SAN by Pitx2c. These observations also

indicate that Pitx2c is a critical factor for the entire left morphogenetic program in the

sinuatrial region. None of the genes studied here were deregulated in other sites of

Pitx2c expression of Pitx2c mutants, including the atrioventricular canal (Tbx3) or

atria (Nkx2-5, Nppa, Cx40) (Figure 6B and data not shown), indicating that Pitx2c

does not directly control expression of these genes. Asymmetry of the venous pole

is first observed after E9.5,26 when the sinus horns are being formed from their pro-

genitors.16 Pitx2 was found to control asymmetric morphogenesis at least in part by

regulation of cell-type specific proliferation.34 It is expressed in the left-sided my-

ocardium from very early stages on and, therefore, may control proliferation of the

left venous pole myocardium as soon as it is recruited from the progenitor pool.

Finally, after E14.5 the nodal gene program becomes confined to the SAN

domain. The sinus horns, at the end of gestation recognizable as the myocardial

sleeves surrounding the right and left caval veins and sinus venarum, gradually switch

to a Cx40-positive, Hcn4-negative atrial gene program (Figure 3C). Nkx2-5 expres-

sion gradually increased toward the end of gestation, indicating that it may be re-

sponsible for the switch to the atrial gene program, although the involvement of other

factors can not be excluded at this point. However, despite the up-regulation of

Nkx2-5 expression in the SAN, Cx40 was not activated here, and Hcn4 was not re-

pressed. Therefore, we hypothesize that in the SAN primordium, Tbx3 expression is,

or has become, insensitive to Nkx2-5, and further that Tbx3 shields the SAN domain

from atrial gene expression and differentiation once Nkx2-5 is being activated in the

SAN (Figure 4C). Although this putative function of Tbx3 remains to be demonstrated,

it is consistent with the found ability of Tbx3 to suppress Nppa and Cx40, and to block

chamber differentiation in the embryonic heart tube.

Acknowledgements

We thank Sameer Rana for his laboratory assistance.


98


Sources of funding

This work was supported by grants from the Netherlands Heart Foundation (96.002

to V.M.C. and A.F.M.M.); the Netherlands Organization for Scientific Research (Vidi

grant 864.05.006 to V.M.C); European Union FP6 contract “Heart Repair” (LSHM-

CT-2005-018630 to V.M.C., A.F.M.M., N.A.B.); the National Health and Medical Re-

search Council (Australia) (354400), the Network for Genes and Environment in

Development (Australia), the National Institute of Child Health and Human Develop-

ment, NIH (HD047858), and the Johnson and Johnson Focused Giving Program (to

R.P.H. and O.W.J.P.); the NIH (RO1 HD33082 to V.E.P.); and the British Heart Foun-

dation (RG RG-03-012 to N.A.B.)

99


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